A shift in dengue cases toward the adult population, accompanied by an increased risk of severe cases of dengue in the elderly, has created an important emerging issue in the past decade. To understand the level of past DENV infection among older adults after a large dengue outbreak occurred in southern Taiwan in 2015, we screened 1498 and 2603 serum samples from healthy residents aged ≥ 40 years in Kaohsiung City and Tainan City, respectively, to assess the seroprevalence of anti-DENV IgG in 2016. Seropositive samples were verified to exclude cross-reaction from Japanese encephalitis virus (JEV), using DENV/JEV-NS1 indirect IgG ELISA. We further identified viral serotypes and secondary DENV infections among positive samples in the two cities. The overall age-standardized seroprevalence of DENV-IgG among participants was 25.77% in Kaohsiung and 11.40% in Tainan, and the seroprevalence was significantly higher in older age groups of both cities. Although the percentages of secondary DENV infection in Kaohsiung and Tainan were very similar (43.09% and 44.76%, respectively), DENV-1 and DENV-2 spanned a wider age range in Kaohsiung, whereas DENV-2 was dominant in Tainan. As very few studies have obtained the serostatus of DENV infection in older adults and the elderly, this study highlights the need for further investigation into antibody status, as well as the safety and efficacy of dengue vaccination in these older populations.